Post-initiation mechanisms, like those regulating the ethanolamine utilization (eut) genes in E. faecalis, are incompletely understood representing a critical gap in knowledge. The long-term goal of this research is to determine how ethanolamine (EA) utilization is regulated in E. faecalis. The objective of this application is to elucidate the post-initiation regulatory mechanisms that activate gene expression. The central hypothesis is that the AmiR and NasR Transcriptional Antiterminator Regulators' (ANTARs) RNA substrates are the central regulatory feature of the system and control gene expression by three interrelated mechanisms. The central hypothesis will be tested in three aims. Aim 1 will elucidate the molecular details of how EutV, the ANTAR in the eut system, interacts with its RNA substrates to control gene expression. There is evidence that EutV binds a dual hairpin RNA structure with specific features. To further understand the structure of this complex, biophysical approaches will be employed, including resolution of three-dimensional structures of the protein-RNA complex by X-ray crystallography. In Aim 2, the purpose of the multiple promoters and terminators in the eut locus will be identified. The working hypothesis is that the transcriptional complexity in the eut locus modulates the levels of both the structural and enzymatic gene products to enable functional EA utilization. Approaches to test this hypothesis will include RNA hybridization, 5'RACE, qRT-PCR, quantitative western and mutational analysis. In Aim 3, the mechanism by which the AdoCbl riboswitch regulates gene expression will be uncovered. The novel, working hypothesis is that a dual hairpin substrate just downstream of the riboswitch binds and sequesters active EutV, preventing induction by EA alone. AdoCbl binding to the riboswitch causes a conformational change that prevents EutV sequestration, allowing for induction when both EA and AdoCbl are present. The model will be assessed by assaying in vivo gene expression following mutation, and by in vitro biochemical approaches to probe RNA structural changes and complex formation. Finally, in silico, bioinformatic analysis will be used to investigate how broadly the findings apply to microbial systems. The research in this proposal will further the understanding of how the eut genes are regulated, contributing knowledge to the field of prokaryotic gene regulation and to the identification of potential antimicrobial targets. Specifically, the significance of this contribution will be the uncovering o novel mechanisms by which ANTARs, riboswitches, and transcriptional terminators control gene expression. The proposed research is innovative because these new mechanisms will challenge the status quo and expand the field's thinking on how post-initiation regulatory features can operate.